CRFG-1a, a target and marker for chronic renal failure

ABSTRACT

CRFG-1a polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CRFG-1a polypeptides and polynucleotides in the design of protocols for the treatment of chronic renal disease, renal ischemia, diabetic nephropathy, acute renal failure, Neurodegenerative disease, and Alzeimer&#39;s disease, among others, and diagnostic assays for such conditions.

This application is a division of application Ser. No. 09/020,466, filedFeb. 9, 1998, now U.S. Pat. No. 5,879,908, which claims the benefit ofU.S. Provisional Application No. 60/045,203, filed Apr. 30, 1997, bothof whose entire contents are incorporated herein by reference in theirentireties. This application claims the benefit of U.S. ProvisionalApplication No. 60/045,203. filed Apr. 30, 1997, which is hereinincorporated by reference in its entirety.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to GTP binding proteinfamily, hereinafter referred to as chronic renal failure gene-1a(CRFG-1a). The invention also relates to inhibiting or activating theaction of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

The sequence of CRFG-1a is similar to uncharacterized putative GTPbinding proteins of yeast (YPL093w), Halobacterium cutirubrum andGTP1/OBG family of GTP binding proteins from Methanobacteriumthermoautotrophicum. GTP binding proteins play important roles inintracellular transport, protein targeting and vesicle fusion.

This indicates that the GTP binding proteins family has an established,proven history as therapeutic targets. Clearly there is a need foridentification and charaterization of further members of GTP bindingprotein family which can play a role in preventing, ameliorating orcorrecting dysfunctions or diseases, including, but not limited to,chronic renal disease, renal ischemia, diabetic nephropathy, acute renalfailure, Neurodegenerative disease, and Azheimer's disease.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to CRFG-1a polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such CRFG-1a polypeptidesand polynucleotides. Such uses include the treatment of chronic renaldisease, renal ischemia, diabetic nephropathy, acute renal failure,Neurodegenerative disease, and Alzheimer's disease, among others. Instill another aspect, the invention relates to methods to identifyagonists and antagonists using the materials provided by the invention,and treating conditions associated with CRFG-1a imbalance with theidentified compounds. Yet another aspect of the invention relates todiagnostic assays for detecting diseases associated with inappropriateCRFG-1a activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"CRFG-1a" refers, among others, generally to a polypeptide having theamino acid sequence set forth in SEQ ID NO:2 or an allelic variantthereof.

"CRFG-1a activity or CRFG-1a polypeptide activity" or "biologicalactivity of the CRFG-1a or CRFG-1a polypeptide" refers to the metabolicor physiologic function of said CRFG-1a including similar activities orimproved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immunogenic activities ofsaid CRFG-1a.

"CRFG-1a gene" refers to a polynucleotide having the nucleotide sequenceset forth in SEQ ID NO: 1 or allelic variants thereof and/or theircomplements.

"Antibodies" as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

"Isolated" means altered "by the hand of man" from the natural state. Ifan "isolated" composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not "isolated," but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is "isolated", as theterm is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotides" include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, "polynucleotide" refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. "Modified" basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,"polynucleotide" embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. "Polynucleotide" also embraces relatively short polynucleotides,often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. "Polypeptide" refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids."Polypeptides" include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslational natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECLLARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., "Analysis for protein modifications and nonproteincofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "ProteinSynthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci(1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polynucleotidecomparisons.

Preferred polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide having at least a 50,60, 70,80, 85, 90, 95, 97 or 100% identity to a polynucleotide referencesequence of SEQ ID NO:1, wherein said reference sequence may beidentical to the sequence of SEQ ID NO:1 or may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5' or 3' terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by thenumerical percent of the respective percent identity and subtractingthat product from said total number of nucleotides in SEQ ID NO:1, or:

    n.sub.n ≦X.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of nucleotide alterations, X_(n) is thetotal number of nucleotides in SEQ ID NO:1, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of X_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may create nonsense, missense orframeshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50,60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said reference sequence may be identical to thesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnonconservative substitution, or insertion, and wherein said alterationsmay occur at the amino- or carboxy-terminal positions of the referencepolypeptide sequence or anywhere between those terminal positions,interspersed either individually among the amino acids in the referencesequence or in one or more contiguous groups within the referencesequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the numerical percent of the respective percent identity andsubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of X_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

Polypeptides of the Invention

In one aspect, the present invention relates to CRFG-1a polypeptides (orCRFG-1a proteins). The CRFG-1a polypeptides include the polypeptide ofSEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acidsequence of SEQ ID NO:2; and polypeptides comprising the amino acidsequence which have at least 80% identity to that of SEQ ID NO:2 overits entire length, and still more preferably at least 90% identity, andeven still more preferably at least 95% identity to SEQ ID NO:2.Furthermore, those with at least 97-99% are highly preferred. Alsoincluded within CRFG-1a polypeptides are polypeptides having the aminoacid sequence which have at least 80% identity to the polypeptide havingthe amino acid sequence of SEQ ID NO:2 over its entire length, and stillmore preferably at least 90% identity, and still more preferably atleast 95% identity to SEQ ID NO:2. Furthermore, those with at least97-99% are highly preferred. Preferably CRFG-1a polypeptide exhibit atleast one biological activity of CRFG-1a.

The CRFG-1a polypeptides may be in the form of the "mature" protein ormay be a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Fragments of the CRFG-1a polypeptides are also included in theinvention. A fragment is a polypeptide having an amino acid sequencethat entirely is the same as part, but not all, of the amino acidsequence of the aforementioned CRFG-1a polypeptides. As with CRFG-1apolypeptides, fragments may be "free-standing," or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof CRFG-1a polypeptide. In this context "about" includes theparticularly recited ranges larger or smaller by several, 5, 4, 3, 2 or1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of CRFG-1a polypeptides, except for deletion ofa continuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Other preferred fragments are biologicallyactive fragments. Biologically active fragments are those that mediateCRFG-1a activity, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Also included arethose that are antigenic or immunogenic in an animal, especially in ahuman.

Preferably, all of these polypeptide fragments retin the biologicalactivity of the CRFG-1a, including antigenic activity. Among the mostpreferred fragment is that having the amino acid sequence of SEQ IDNO:4. Variants of the defined sequence and fragments also form part ofthe present invention. Preferred variants are those that vary from thereferents by conservative amino acid substitutions--i.e., those thatsubstitute a residue with another of like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tvr. Particularlypreferred are variants in which several, 5-10, 1-5, or 1-2 amino acidsare substituted, deleted, or added in any combination.

The CRFG-1a polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

Another aspect of the invention relates to CRFG-1a polynucleotides.CRFG-1a polynucleotides include isolated polynucleotides which encodethe CRFG-1a polypeptides and fragments, and polynucleotides closelyrelated thereto. More specifically, CRFG-1a polynucleotide of theinvention include a polynucleotide comprising the nucleotide sequencecontained in SEQ ED NO:1 encoding a CRFG-1a polypeptide of SEQ ID NO:2,and polynucleotides having the particular sequences of SEQ ID NOS:1 and3. CRFG-1a polynucleotides further include a polynucleotide comprising anucleotide sequence that has at least 80% identity over its entirelength to a nucleotide sequence encoding the CRFG-1a polypeptide of SEQID NO:2, and a polynucleotide comprising a nucleotide sequence that isat least 80% identical to that of SEQ ID NO:1 over its entire length. Inthis regard, polynucleotides at least 90% identical are particularlypreferred, and those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred and those withat least 98-99% are most highly preferred, with at least 99% being themost preferred. Also included under CRFG-1a polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO:1 to hybridize under conditions useablefor amplification or for use as a probe or marker. The invention alsoprovides polynucleotides which are complementary to such CRFG-1apolynucleotides.

CRFG-1a of the invention is structurally related to other proteins ofthe GTP binding protein family, as shown by the results of sequencingthe cDNA encoding human CRFG-1a. The cDNA sequence of SEQ ID NO:1contains an open reading frame (nucleotide number 3 to 1904) encoding apolypeptide of 634 amino acids of SEQ ID NO:2. The amino acid sequenceof Table 1 (SEQ ID NO:2) has about 46.1% identity (using FASTA) in 634amino acid residues with Hypothetical protein YPL093w from yeast (H.Bussey et al. Nature 387 (6632 Suppl), 103-105, 1997). The nucleotidesequence of Table 1 (SEQ ID NO:1) has about 59.7% identity (using FASTA)in 1351 nucleotide residues with Saccharomyces cerevisiae chromosome XVIcosmid 8059/8047 (H. Bussey et al. Nature 387 (6632 Suppl), 103-105,1997). Thus, CRFG-1a polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides, and their utility is obvious to anyone skilled in theart.

                  TABLE 1.sup.a                                                   ______________________________________                                                                      AGCATGGCACATTACAACTTCAAGAAAATTACGGTGGTGC40                                     - CGTCCGCCAAGGACTTCATAGACCTCACGTTGTCCAAGAC8                                 0                                                   - TCAACGAAAGACTCCAACCGTTATTCATAAACATTACCAA120                                 - ATACATCGCATTAGACATTTTTACATGAGAAAAGTCAAAT160                                 - TTACTCAACAGAATTACCATGATAGACTTTCACAAATTCT200                                 - AACAGATTTCCCCAAATTGGATGATATTCATCCGTTCTAT240                                 - GCTGATTTGATGAATATTCTCTACGACAAGGATCATTACA280                                 - AGTTGGCTCTGGGGCAAATAAATATTGCCAAAAATTTAGT320                                 - GGACAATGTTGCTAAAGATTATGTGCGACTGATGAAGTAT360                                 - GGCGACTCTCTCTACCGCTGCAAACAGCTGAAGCGTGCGG400                                 - CCCTGGGACGGATGTGCACAGTGATCAAGAGGCAGAAGCA440                                 - GAGTTTGGAGTATTTGGAGCAAGTGCGTCAGCATTTATCC480                                 - CGTTTGCCAACCATTGATCCGAATACCAGGACCCTGCTTT520                                 - TGTGTGGGTACCCAAATGTTGGGAAGTCCAGCTTCATCAA560                                 - CAAGGTGACGAGAGCAGACGTGGATGTCCAGCCCTATGCG600                                 - TTCACAACCAAGTCTCTGTTTGTTGGGCACATGGATTATA640                                 - AGTATCTACGTTGGCAGGTTGTAGACACTCCTGGGATCCT680                                 - GGACCACCCTCTGGAGGATAGGAACACCATCGAGATGCAG720                                 - GCCATCACTGCCCTGGCCCACCTCCGTGCTGCGGTCCTGT760                                 - ATGTGATGGATTTGTCTGAGCAGTGTGGGCATGGGCTGAG800                                 - GGAACAGCTAGAACTCTTCCAGAACATCAGACCTCTCTTC840                                 - ATCAACAAGCCTCTCATAGTTGTAGCCAACAAATGTGATG880                                 - TGAAGAGAATAGCTGAACTTTCTGAAGATGATCAGAAAAT920                                 - ATTTACAGATTTGCAGTCTGAAGGATTCCCTGTAATAGAG960                                 - ACCAGCACCCTGACTGAGGAAGGTGTTATTAAAGTTAAAA1000                                - CAGAGGCTTGCGATAGGCTTTTGGCTCATCGAGTGGAAAC1040                                - CAAAATGAAGGGAAATAAAGTGAATGAGGTGCTGAATAGA1080                                - CTGCACCTGGCTATCCCAACCAGGAGGGACGATAAGGAGA1120                                - GGCCCCCTTTCATCCCTGAAGGAGTGGTGGCTCGCAGGAA1160                                - GAGGATGGAAACTGAGGAGTCCAGGAAGAAGAGGGAACGA1200                                - GATCTTGAGCTGGAAATGGGAGATGATTATATTTTGGATC1240                                - TTCAGAAGTACTGGGATTTAATGAATTTGTCTGAAAAACA1280                                - TGATAAGATACCAGAAATCTGGGAAGGCCATAATATAGCT1320                                - GATTATATTGATCCAGCCATCATGAAGAAATTGGAAGAAT1360                                - TAGAAAAAGAAGAAGAGCTGAGAACAGCTGCTGGAGAGTA1400                                - TGACAGTGTATCTGAGAGTGAAGACGAAGAGATGCTGGAA1440                                - ATCCGACAGCTGGCAAAGCAAATTCGAGAGAAAAAGAAGT1480                                - TGAAAATTCTGGAGTCCAAAGAAAAGAATACACAGGGACC1520                                - CAGGATGCCGCGAACTGCTAAGAAGGTTCAGAGGACAGTT1560                                - TTGGAGAAGGAGATGCGTAGTCTTGGTGTTGACATGGACG1600                                - ATAAAGACGATGCCCATTACGCAGTCCAGGCAAGAAGATC1640                                - CCGGAGCATCACTAGGAAAAGAAAGCGGGAAGACTCTGCT1680                                - CCCCCGTCCTCTGTGGCCCGGAGTGGGAGTTGCTCTCGAA1720                                - CTCCACGTGACGTTTCTGGTCTTAGGGATGTCAAGATGGT1760                                - GAAGAAAGCCAAGACTATGATGAAGAATGCTCAGAAGAAG1800                                - ATGAATCGGTTGGGGAAGAAAGGGGAGGCGGATAGACACG1840                                - TGTTTGATATGAAGCCCAAGCACTTGCTGTCTGGGAAGAG1880                                - GAAAGCTGGTAAAAAGGACAGGAGATAGTATCCGTTTGGT1920                                - TGGCGTGGCTTCGCTAGAGTGTTGCTGTTTATTTCCTGTT1960                                - TTGGCACAGTATGGTTTCATGAAATTGGAGCTCTGTATAA2000                                - ACTGAAAAAGACAAAATAAGTAAAGCACTTGTTGCTTTGC2040                                - TGAAAACTATGGTTAACCCTATATAGGTGTGGGAAATTTT2080                                - TGTCACTGCATAATATTACAAATATTTTGAGTAGACAGTG2120                                - TTTCCACATTTAATGGAGTATCAGTTGCTTCAGATTTTCA2160                                - GAACTGGGAAGATTTACTGGTGTAACTGGGTTGTTTTTGA2200                                - TGGAGAAAAACCTTATTTTCTTTTGTAAGAGCTGGGAGCA2240                                - AACACGTTTATGAGTGTGTCGGAATCCCGTGCTTAAAATA2280                                - CGCTCTTAAATTATTTTCTAGTCTTATTTCACAATGTCTC2320                                - ATTGTAGTCTGTCTTCAACTATTTTATCCAAAATANACCT2360                                - CCAGAAGAAAG2371                                                          ______________________________________                                         .sup.a A nucleotide sequence of a human CRFG1a(SEQ ID NO: 1).            

                  TABLE 2.sup.b                                                   ______________________________________                                          MAHYNFKKITVVPSAKDFIDLTLSKTQRKTPTVIHKHYQI40                                     - HRIRHFYMRKVKFTQQNYHDRLSQILTDFPKLDDIHPFYA80                                  - DLMNILYDKDHYKLALGQINIAKNLVDNVAKDYVRLMKYG120                                 - DSLYRCKQLKRAALGRMCTVIKRQKQSLEYLEQVRQHLSR160                                 - LPTIDPNTRTLLLCGYPNVGKSSFINKVTRADVDVQPYAF200                                 - TTKSLFVGHMDYKYLRWQVVDTPGILDHPLEDRNTIEMQA240                                 - ITALAHLRAAVLYVMDLSEQCGHGLREQLELFQNIRPLFI280                                 - NKPLIVVANKCDVKRIAELSEDDQKIFTDLQSEGFPVIET320                                 - STLTEEGVIKVKTEACDRLLAHRVETKMKGNKVNEVLNRL360                                 - HLAIPTRRDDKERPPFIPEGVVARRKRMETEESRKKRERD400                                 - LELEMGDDYILDLQKYWDLMNLSEKHDKIPEIWEGHNIAD440                                 - YIDPAIMKKLEELEKEEELRTAAGEYDSVSESEDEEMLEI480                                 - RQLAKQIREKKKLKILESKEKNTQGPRMPRTAKKVQRTVL520                                 - EKEMRSLGVDMDDKDDAHYAVQARRSRSITRKRKREDSAP560                                 - PSSVARSGSCSRTPRDVSGLRDVKMVKKAKTMMKNAQKKM600                                 - NRLGKKGEADRRVFDMKPKHLLSGKRKAGKKDRR634                                    ______________________________________                                         .sup.b An amino acid sequence of a human CRFGla (SEQ ID NO: 2).          

One polynucleotide of the present invention encoding CRFG-1a may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human kidney and testes using theexpressed sequence tag (EST) analysis (Adams, M. D., etal. Science(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992)355:632-634;Adams, M.D., et al, Nature (1995)377 Supp:3-174). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

The nucleotide sequence encoding CRFG-1a polypeptide of SEQ ID NO:2 maybe identical to the polypeptide encoding sequence contained in Table 1(nucleotide number 3 to 1904 of SEQ ID NO:1), or it may be a sequence,which as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinantproduction of CRFG-1 a polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro-proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc NatlAcad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide mayalso contain non-coding 5' and 3' sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding CRFG-1avariants comprising the amino acid sequence of CRFG-1a polypeptide ofTable 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacid residues are substituted, deleted or added, in any combination.Among the preferred polynucleotides of the present invention iscontained in Table 3 (SEQ ID NO:3) encoding the amino acid sequence ofTable 4 (SEQ ID NO:4).

                  TABLE 3.sup.c                                                   ______________________________________                                          gACTCTGCTC CCCCGTCCTC TGTGGCCCGG AgTGGGAGTT                                    - GCTCTCGAAC TCCACGTGAC GTTTCTGGTC TTAGGGATGT80                               - CAAgATGGTG AAgAAAGCCA AGACTATGAT GAAGAATGCT                                 - CAgAAgAAgA TGAATCGGTT GGGGAAgAAA GGGGAGGCGG160                              - ATATACACTT gTTTGATATG AAGCCCAAgC ACTTGCTGTC                                 - TGGGAAgAGG AAAGCTGGTa AAAAGGACAG GAGATAgTAT240                              - CCGTTTGGTT GGCGTGGCTT CGCTAgAgTG TTGCTGTTTA                                 - TTTCCTGGTT TGGCACAGTA TGGTTTCaTG AAATTGGAGC320                              - TCTGTaTAAA CTGAAAAAGA CAAAATAAGT AAAGCACTTG                                 - TTGCTTTGCT GAAAACTATG GTTAACCCTA TATAGGTGTG400                              - GGAAATTTTT GTCaCTGCAT AATATTACaA ATATTCTGAG                                 - TAGACAGtGT TTCCACATTT AATGGAGTAT CAGTTGCTTC480                              - AGATTTTCAG AACTGGGAAG ATTTACTGGT GTAACTGGGT                                 - TGTTTTTGAT GGAGAAAAAC CTTATTTTCT TTTGTAAGAG560                              - CTGGGAGCAA ACACGTTTAT GAGTGTGTCG GAATCCCGTG                                 - CTTAAAATAC GCTCTTAAAT tATTTTCTAG TCCTTATTTT640                              - ACAATGTCTC ATTGTAGTCT GTCTTCAACT ATTTTATCCA                                 - AAATAAACCT CCAGAAGGAA AAAAAAAAAA AAAAAA716                               ______________________________________                                         .sup.c A partial nucleotide sequence of a human CRFG1a(SEQ ID NO: 3).    

                  TABLE 4.sup.d                                                   ______________________________________                                          HEDKDDAHYAVQARRSRSITRKRKREDSAPPSSVARSGSC40                                    SRTPRDVSGLRDVKMVKKAKTMMKNAQKKMNRLGKKGEAD80                                    IHLFDMKPKHLLSGKRKAGKKDRR104                                                 ______________________________________                                         .sup.d A partial amino acid sequence of a human CRFGla(SEQ ID NO: 4).    

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 80%, and preferably at least 90%, andmore preferably at least 95%, yet even more preferably 97-99% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1 or afragment thereof (including that of SEQ ID NO:3), may be used ashybridization probes for cDNA and genomic DNA, to isolate full-lengthcDNAs and genomic clones encoding CRFG-1a polypeptide and to isolatecDNA and genomic clones of other genes (including genes encodinghomologs and orthologs from species other than human) that have a highsequence similarity to the CRFG-1a gene. Such hybridization techniquesare known to those of skill in the art. Typically these nucleotidesequences are 80% identical, preferably 90% identical, more preferably95% identical to that of the referent. The probes generally willcomprise at least 15 nucleotides. Preferably, such probes will have atleast 30 nucleotides and may have at least 50 nucleotides. Particularlypreferred probes will range between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding CRFG-1apolypeptide, including homologs and orthologs from species other thanhuman, comprises the steps of screening an appropriate library understingent hybridization conditions with a labeled probe having the SEQ IDNO:1 or a fragment thereof (including that of SEQ ID NO:3), andisolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothose of skill in the art. Thus in another aspect, CRFG-1apolynucleotides of the present invention further include a nucleotidesequence comprising a nucleotide sequence that hybridize under stringentcondition to a nucleotide sequence having SEQ ID NO:1 or a fragmentthereof (including that of SEQ ID NO:3). Also included with CRFG-1apolypeptides are polypeptide comprising amino acid sequence encoded bynucleotide sequence obtained by the above hybridization condition.Stringent hybridization conditions are as defined above or.alternatively, conditions under overnight incubation at 42° C. in asolution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution. 10%dextran sulfate, and 20 microgram/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dexran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the CRFG-1a polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If CRFG-1a polypeptide is secreted intothe medium, the medium can be recovered in order to recover and purifythe polypeptide; if produced intracellularly, the cells must first belysed before the polypeptide is recovered. CRFG-1a polypeptides can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of CRFG-1a polynucleotides foruse as diagnostic reagents. Detection of a mutated form of CRFG-1a geneassociated with a dysfunction will provide a diagnostic tool that canadd to or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression, over-expression or alteredexpression of CRFG-1a. Individuals canying mutations in the CRFG-1a genemay be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled CRFG-1a nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al,Science (1985) 230:1242. Sequence changes at specific locations may alsobe revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method. See Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401. In another embodiment, an array ofoligonucleotides probes comprising CRFG-1a nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to chronic renal disease, renal ischemia, diabeticnephropathy, acute renal failure, Neurodegenerative disease. andAlzheimer's disease through detection of mutation in the CRFG-1a gene bythe methods described.

In addition, chronic renal disease, renal ischemia, diabeticnephropathy, acute renal failure, Neurodegenerative disease, andAlzheimer's disease, can be diagnosed by methods comprising determiningfrom a sample derived from a subject an abnormally decreased orincreased level of CRFG-1a polypeptide or CRFG-1a mRNA. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods. Assay techniques that can be used todetermine levels of a protein, such as an CRFG-1a polypeptide, in asample derived from a host are well-known to those of skill in the art.Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostickit for a disease or suspectability to a disease, particularly chronicrenal disease, renal ischemia, diabetic nephropathy, acute renalfailure, Neurodegenerative disease, and Atzheimer's disease, whichcomprises:

(a) a CRFG-1a polynucleotide, preferably the nucleotide sequence of SEQID NO:1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a CRFG-1a polypeptide, preferably the polypeptide of SEQ ID NO:2, ora fragment thereof; or

(d) an antibody to a CRFG-1a polypeptide, preferably to the polypeptideof SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b),(c) or (d) may comprise a substantial component.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thedifferences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

The CRFG-1a gene (SEQ ID NO:1) is found on chromosome 10p15.2-15.3 whichis associated with glioma of the brain.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the CRFG-1a polypeptides. The term"immunospecific" means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the CRFG-1a polypeptides can be obtained byadministering the polypeptides or epitope-bearing fragments, analogs orcells to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and theEBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCERTHERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against CRFG-1a polypeptides may also be employed to treatchronic renal disease, renal ischemia, diabetic nephropathy, acute renalfailure, Neurodegenerative disease, and Alzheimer's disease, amongothers.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with CRFG-1a polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom chronic renal disease, renal ischemia, diabetic nephropathy, acuterenal failure, Neurodegenerative disease, and Alzheimer's disease, amongothers. Yet another aspect of the invention relates to a method ofinducing immunological response in a mammal which comprises, deliveringCRFG-1a polypeptide via a vector directing expression of CRFG-1apolynucleotide in vivo in order to induce such an immunological responseto produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a CRFG-1apolypeptide wherein the composition comprises a CRFG-1a polypeptide orCRFG-1a gene. The vaccine formulation may further comprise a suitablecarrier. Since CRFG-1a polypeptide may be broken down in the stomach, itis preferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradernal etc. injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The CRFG-1a polypeptide of the present invention may be employed in ascreening process for compounds which activate (agonists) or inhibitactivation of (antagonists, or otherwise called inhibitors) the CRFG-1apolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess identify agonist or antagonistsfrom, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. These agonists or antagonists may benatural or modified substrates, ligands, enzymes, receptors, etc., asthe case may be, of the polypeptide of the present invention; or may bestructural or functional mimetics of the polypeptide of the presentinvention. See Coligan et al., Current Protocols in Immunology1(2):Chapter5 (1991).

CRFG-1a polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate CRFG-1a polypeptide on the one handand which can inhibit the function of CRFG-1a polypeptide on the otherhand. In general, agonists are employed for therapeutic and prophylacticpurposes for such conditions as chronic renal disease, renal ischemia,diabetic nephropathy, acute renal failure, Neurodegenerative disease,and Alzheimer's disease. Antagonists may be employed for a variety oftherapeutic and prophylactic purposes for such conditions as chronicrenal disease, renal ischernia, diabetic nephropathy, acute renalfailure, Neurodegenerative disease, and Alzheimer's disease.

In general, such screening procedures may involve using appropriatecells which express the CRFG-1a polypeptide or respond to CRFG-1apolypeptide of the present invention. Such cells include cells frommammals, yeast, Drosophila or E. coli. Cells which express the CRFG-1apolypeptide (or cell membrane containing the expressed polypeptide) orrespond to CRFG-1a polypeptide are then contacted with a test compoundto observe binding, or stimulation or inhibition of a functionalresponse. The ability of the cells which were contacted with thecandidate compounds is compared with the same cells which were notcontacted for CRFG-1a activity.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the CRFG-1a polypeptide is detected bymeans of a label directly or indirectly associated with the candidatecompound or in an assay involving competition with a labeled competitor.Further, these assays may test whether the candidate compound results ina signal generated by activation of the CRFG-1a polypeptide, usingdetection systems appropriate to the cells bearing the CRFG-1apolypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed.

Further, the assays may simply comprise the steps of mixing a candidatecompound with a solution containing a CRFG-1a polypeptide to form amixture, measuring CRFG-1a activity in the mixture, and comparing theCRFG-1a activity of the mixture to a standard.

The CRFG-1a cDNA, protein and antibodies to the protein may also be usedto configure assays for detecting the effect of added compounds on theproduction of CRFG-1a mRNA and protein in cells. For example, an ELISAmay be constructed for measuring secreted or cell associated levels ofCRFG-1a protein using monoclonal and polyclonal antibodies by standardmethods known in the art, and this can be used to discover agents whichmay inhibit or enhance the production of CRFG-1a (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

The CRFG-1a protein may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the CRFG-1a is labeled with a radioactiveisotope (eg 125I), chemically modified (eg biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (cells, cell membranes, cellsupernatants, tissue extracts, bodily fluids). Other methods includebiophysical techniques such as surface plasmon resonance andspectroscopy. In addition to being used for purification and cloning ofthe receptor, these binding assays can be used to identify agonists andantagonists of CRFG-1a which compete with the binding of CRFG-1a to itsreceptors, if any. Standard methods for conducting screening assays arewell understood in the art.

Examples of potential CRFG-1a polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, enzymes, receptors, etc., as thecase may be, of the CRFG-1a polypeptide, e.g., a fragment of theligands, substrates, enzymes, receptors, etc.; or small molecules whichbind to the polypeptide of the present invention but do not elicit aresponse, so that the activity of the polypeptide is prevented.

Thus in another aspect, the present invention relates to a screening kitfor identifying agonists, antagonists, ligands, receptors, substrates,enzymes, etc. for CRFG-1a polypeptides; or compounds which decrease orenhance the production of CRFG-1a polypeptides, which comprises:

(a) a CRFG-1a polypeptide, preferably that of SEQ ID NO:2;

(b) a recombinant cell expressing a CRFG-1a polypeptide, preferably thatof SEQ ID NO:2;

(c) a cell membrane expressing a CRFG-1a polypeptide; preferably that ofSEQ ID NO:2: or

(d) antibody to a CRFG-1a polypeptide, preferably that of SEQ ID NO:2.It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as,chronic renal disease, renal ischemia, diabetic nephropathy, acute renalfailure, Neurodegenerative disease, and Alzheimer's disease, related toboth an excess of and insufficient amounts of CRFG-1a polypeptideactivity.

If the activity of CRFG-1a polypeptide is in excess, several approachesare available. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitthe function of the CRFG-1a polypeptide, such as, for example, byblocking the binding of ligands, substrates, enzymes, receptors, etc.,or by inhibiting a second signal, and thereby alleviating the abnormalcondition. In another approach, soluble forms of CRFG-1a polypeptidesstill capable of binding the ligand, substrate, enzymes, receptors, etc.in competition with endogenous CRFG-1a polypeptide may be administered.Typical embodiments of such competitors comprise fragments of theCRFG-1a polypeptide.

In another approach, soluble forms of CRFG-1a polypeptides still capableof binding the ligand in competition with endogenous CRFG-1a polypeptidemay be administered. Typical embodiments of such competitors comprisefragments of the CRFG-1a polypeptide.

In still another approach, expression of the gene encoding endogenousCRFG-1a polypeptide can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O'Connor, J Neurochem (1991) 56:560 inOlizodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied. See, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988)241:456; Dervan et al., Science (1991) 251:1360. These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression ofCRFG-1a and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates CRFG-1a polypeptide,i.e., an agonist as described above, in combination with apharmaceutically acceptable carrier, to thereby alleviate the abnormalcondition. Alternatively, gene therapy may be employed to effect theendogenous production of CRFG-1a by the relevant cells in the subject.For example, a polynucleotide of the invention may be engineered forexpression in a replication defective retroviral vector, as discussedabove. The retroviral expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding a polypeptide of the present inventionsuch that the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo. For overview of gene therapy, see Chapter20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Anotherapproach is to administer a therapeutic amount of CRFG-1a polypeptidesin combination with a suitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of CRFG-1a polypeptides, and agonistsand antagonist peptides or small molecules, may be formulated incombination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof Formulationshould suit the mode of administration, and is well within the skill ofthe art. The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as "gene therapy" asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

Two animal model systems have been studied to provide targets forintervention in chronic renal failure. CRFG-1a is a novel geneidentified by differential display PCR to be down regulated in threeanimal models of chronic renal failure, the obese Zucker rat and the 5/6nephrectomized rat. In addition, aged Fisher 344 rats (24 months old),which have enlarged kidneys and reduced renal function, also havedecreased expression of CRFG-1a. Loss of expression of this gene inrenal failure may indicate an important role in normal functioning ofthe kidney.

In 5-month old obese Zucker rats, that have developed chronic renalfailure, CRFG-1a mRNA is decreased to less than 1/3 of levels seen inlean and healthy age-matched controls. Because there is no correlationof proteinuria with CRFG-1a expression, a decrease in CRFG-1a is anearly marker of renal impairment before standard clinical indicators.

While in the rat, CRFG-1a has 2 mRNA sizes of 2.5 and 1.5 kb, in thehuman only one molecular weight species is identified at 2.7 kb. HumanCRFG-1a was mapped to human chromosome 10p15.2-15. This region ofchromosome 10 is linked to 171840 PHOSPHOFRUCTOKINASE, PLATELET TYPE,600449 DIHYDRODIOL DEHYDROGENASE, TYPE I; 601070 INTERLEUKIN-15RECEPTOR, ALPHA; 600448 PROTEIN KINASE C, THETA FORM; 147270INTER-ALPHA-TRYPSIN INHIBITOR, HEAVY CHAIN-1; 176870 PROTEIN HC; 137800GLIOMA OF BRAIN, 300007 INTERLEUKIN-9 RECEPTOR; and 147680INTERLEUKIN-2.

Normal mRNA expression on a multiple tissue northern blot identifiesexpression in skeletalmuscle>testes>pancreas>heart>thymus>placenta>kidney>leukocytes>spleen>ovary>colon>brain>prostate.

EXAMPLE 1:

The existence of CRFG-1a was determined by differential displaypolymerase chain reaction (DDPCR) as developed by Liang P. and Pardee A.B. Science. Aug. 14, 1992; Volume 257, pages 967-71. An oligonucleotideprimer (Primer I), 5'-ACCACACATCTGA-3' (SEQ ID NO:5) was used in thesynthesis of complementary DNA (cDNA) from RNA of lean and obese Zuckerrat kidneys. cDNA was synthesized in a 20 μl volume with 0.5 μg RNA, 1μM primer I, 20 μM dNTP, 400 units M-MLV reverse transcriptase (Promega,Madison, Wis.), and 1× standard reverse transcriptase buffer as given bythe manufacturer. The reaction sample was heated to 65 degrees C. for 5minutes and then incubated at 42 degrees C. for one hour. Polymerasechain reaction (PCR) was then performed in a 20 μl reaction volume using4 μl of the cDNA synthesis reaction from above with 1.5 mM MgCl₂, 20 μMDNTP, 1 μM primer I, 1 μM primer II (5'-TGTTGGGAACAAG -3') (SEQ IDNO:6), 2 μCi [33-P]-d-alpha-ATP, 1.25 U Amplitaq polymerase (PerkinElmer, Foster City, Calif.) and 1× standard PCR buffer from themanufacturer. PCR cycling conditions were 40 cycles of 94° C. for 30seconds, 42° C. for 2 minutes, 72° C. for 30 seconds with a finalextension of 72° C. for 5 minutes. Labeled PCR fragments from lean andobese Zucker rat Kidney RNA were resolved on a 12% SDS-polyacrylamidegel and exposed to X-ray film for 16 hours. A PCR amplified DNA fragmentof 225 nucleotides was identified to be decreased in obese Zucker ratkidneys compared to lean age matched control rats. The fragment wasexcised from the dried polyacrylamide gel and DNA was eluted withboiling water. The eluted DNA was subjected to PCR using the sameconditions as above. A 2 μl aliquot of the PCR reaction was then used tosubclone the PCR fragment into the pCRII vector (Invitrogen, Carlsbad,Calif.) using standard reaction conditions of the manufacturer. The cDNAinsert was then sequenced with the fmol sequencing kit (Promega,Madison, Wis.).

The sequence information was used to generate an anti-sense primer forthe cloning of additional 5' sequence using the Marathon RACE kit(Clonetech, Palo, Alto, Calif.). The 607 nucleotide sequence obtainedfrom the Marathon RACE kit was then used to identify the human homologueusing the BLAST sequence analysis algorithm. The full length human cDNAwas cloned from kidney mRNA using the Marathon Race kit. The humansequence is given in SEQ ID NO:1.

EXAMPLE 2

CRFG-1a mRNA was detected by northern blot in RNA from rat kidneys.Total RNA was extracted from renal cortex by guanidinium thiocyanatedenaturation and acidified phenol-chloroform extraction (CHOMCZYNSKI P,SACCHI N: Analyt Biochem 162:156-159, 1987). Total RNA (10 μg) wasfractionated on 0.2M formaldehyde- 1% agarose gels and transferred tonylon membranes (Nylon-1, Gibco-BRL, Gaithersburg, Md.) in 4× standardsaline citrate. Equivalent loading and transfer were verified bymethylene blue staining. Antisense [32P]cDNA probes were made forCRFG-1a that recognizes mRNA from rat at 1.5 and 2.5 kb. Northern blotanalysis showed that CRFG-1a mRNA was decreased in kidneys from obeseZucker rats which develop renal failure. CRFG-1a mRNA was also decreasedin kidneys after partial nephrectomy where 5/6 of the total renal masswas removed. This animal model develops chronic renal disease (Shea S M,Raskova J, Morrison AB Am J Pathol 1980 Aug; 100(2):513-528 ). CRFG-1amRNA was also decreased in kidneys of aging F344 rats. F344 rats developrenal disease with advancing age (McDermott G F, Ingram A, Scholey J,Kirkland JL, Whiteside C I J Gerontol A Biol Sci Med Sci 1996 Mar;51(2):M80-M85), suggesting that CRFG-1a is decreased in renal disease.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2371 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AGCATGGCAC ATTACAACTT CAAGAAAATT ACGGTGGTGC CGTCCGCCAA GG -            #ACTTCATA     60                                                                 - - GACCTCACGT TGTCCAAGAC TCAACGAAAG ACTCCAACCG TTATTCATAA AC -            #ATTACCAA    120                                                                 - - ATACATCGCA TTAGACATTT TTACATGAGA AAAGTCAAAT TTACTCAACA GA -            #ATTACCAT    180                                                                 - - GATAGACTTT CACAAATTCT AACAGATTTC CCCAAATTGG ATGATATTCA TC -            #CGTTCTAT    240                                                                 - - GCTGATTTGA TGAATATTCT CTACGACAAG GATCATTACA AGTTGGCTCT GG -            #GGCAAATA    300                                                                 - - AATATTGCCA AAAATTTAGT GGACAATGTT GCTAAAGATT ATGTGCGACT GA -            #TGAAGTAT    360                                                                 - - GGCGACTCTC TCTACCGCTG CAAACAGCTG AAGCGTGCGG CCCTGGGACG GA -            #TGTGCACA    420                                                                 - - GTGATCAAGA GGCAGAAGCA GAGTTTGGAG TATTTGGAGC AAGTGCGTCA GC -            #ATTTATCC    480                                                                 - - CGTTTGCCAA CCATTGATCC GAATACCAGG ACCCTGCTTT TGTGTGGGTA CC -            #CAAATGTT    540                                                                 - - GGGAAGTCCA GCTTCATCAA CAAGGTGACG AGAGCAGACG TGGATGTCCA GC -            #CCTATGCG    600                                                                 - - TTCACAACCA AGTCTCTGTT TGTTGGGCAC ATGGATTATA AGTATCTACG TT -            #GGCAGGTT    660                                                                 - - GTAGACACTC CTGGGATCCT GGACCACCCT CTGGAGGATA GGAACACCAT CG -            #AGATGCAG    720                                                                 - - GCCATCACTG CCCTGGCCCA CCTCCGTGCT GCGGTCCTGT ATGTGATGGA TT -            #TGTCTGAG    780                                                                 - - CAGTGTGGGC ATGGGCTGAG GGAACAGCTA GAACTCTTCC AGAACATCAG AC -            #CTCTCTTC    840                                                                 - - ATCAACAAGC CTCTCATAGT TGTAGCCAAC AAATGTGATG TGAAGAGAAT AG -            #CTGAACTT    900                                                                 - - TCTGAAGATG ATCAGAAAAT ATTTACAGAT TTGCAGTCTG AAGGATTCCC TG -            #TAATAGAG    960                                                                 - - ACCAGCACCC TGACTGAGGA AGGTGTTATT AAAGTTAAAA CAGAGGCTTG CG -            #ATAGGCTT   1020                                                                 - - TTGGCTCATC GAGTGGAAAC CAAAATGAAG GGAAATAAAG TGAATGAGGT GC -            #TGAATAGA   1080                                                                 - - CTGCACCTGG CTATCCCAAC CAGGAGGGAC GATAAGGAGA GGCCCCCTTT CA -            #TCCCTGAA   1140                                                                 - - GGAGTGGTGG CTCGCAGGAA GAGGATGGAA ACTGAGGAGT CCAGGAAGAA GA -            #GGGAACGA   1200                                                                 - - GATCTTGAGC TGGAAATGGG AGATGATTAT ATTTTGGATC TTCAGAAGTA CT -            #GGGATTTA   1260                                                                 - - ATGAATTTGT CTGAAAAACA TGATAAGATA CCAGAAATCT GGGAAGGCCA TA -            #ATATAGCT   1320                                                                 - - GATTATATTG ATCCAGCCAT CATGAAGAAA TTGGAAGAAT TAGAAAAAGA AG -            #AAGAGCTG   1380                                                                 - - AGAACAGCTG CTGGAGAGTA TGACAGTGTA TCTGAGAGTG AAGACGAAGA GA -            #TGCTGGAA   1440                                                                 - - ATCCGACAGC TGGCAAAGCA AATTCGAGAG AAAAAGAAGT TGAAAATTCT GG -            #AGTCCAAA   1500                                                                 - - GAAAAGAATA CACAGGGACC CAGGATGCCG CGAACTGCTA AGAAGGTTCA GA -            #GGACAGTT   1560                                                                 - - TTGGAGAAGG AGATGCGTAG TCTTGGTGTT GACATGGACG ATAAAGACGA TG -            #CCCATTAC   1620                                                                 - - GCAGTCCAGG CAAGAAGATC CCGGAGCATC ACTAGGAAAA GAAAGCGGGA AG -            #ACTCTGCT   1680                                                                 - - CCCCCGTCCT CTGTGGCCCG GAGTGGGAGT TGCTCTCGAA CTCCACGTGA CG -            #TTTCTGGT   1740                                                                 - - CTTAGGGATG TCAAGATGGT GAAGAAAGCC AAGACTATGA TGAAGAATGC TC -            #AGAAGAAG   1800                                                                 - - ATGAATCGGT TGGGGAAGAA AGGGGAGGCG GATAGACACG TGTTTGATAT GA -            #AGCCCAAG   1860                                                                 - - CACTTGCTGT CTGGGAAGAG GAAAGCTGGT AAAAAGGACA GGAGATAGTA TC -            #CGTTTGGT   1920                                                                 - - TGGCGTGGCT TCGCTAGAGT GTTGCTGTTT ATTTCCTGTT TTGGCACAGT AT -            #GGTTTCAT   1980                                                                 - - GAAATTGGAG CTCTGTATAA ACTGAAAAAG ACAAAATAAG TAAAGCACTT GT -            #TGCTTTGC   2040                                                                 - - TGAAAACTAT GGTTAACCCT ATATAGGTGT GGGAAATTTT TGTCACTGCA TA -            #ATATTACA   2100                                                                 - - AATATTTTGA GTAGACAGTG TTTCCACATT TAATGGAGTA TCAGTTGCTT CA -            #GATTTTCA   2160                                                                 - - GAACTGGGAA GATTTACTGG TGTAACTGGG TTGTTTTTGA TGGAGAAAAA CC -            #TTATTTTC   2220                                                                 - - TTTTGTAAGA GCTGGGAGCA AACACGTTTA TGAGTGTGTC GGAATCCCGT GC -            #TTAAAATA   2280                                                                 - - CGCTCTTAAA TTATTTTCTA GTCTTATTTC ACAATGTCTC ATTGTAGTCT GT -            #CTTCAACT   2340                                                                 - - ATTTTATCCA AAATANACCT CCAGAAGAAA G        - #                  - #            2371                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 634 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ala His Tyr Asn Phe Lys Lys Ile Thr Va - #l Val Pro Ser Ala Lys       1               5  - #                10  - #                15               - - Asp Phe Ile Asp Leu Thr Leu Ser Lys Thr Gl - #n Arg Lys Thr Pro Thr                  20      - #            25      - #            30                   - - Val Ile His Lys His Tyr Gln Ile His Arg Il - #e Arg His Phe Tyr Met              35          - #        40          - #        45                       - - Arg Lys Val Lys Phe Thr Gln Gln Asn Tyr Hi - #s Asp Arg Leu Ser Gln          50              - #    55              - #    60                           - - Ile Leu Thr Asp Phe Pro Lys Leu Asp Asp Il - #e His Pro Phe Tyr Ala      65                  - #70                  - #75                  - #80        - - Asp Leu Met Asn Ile Leu Tyr Asp Lys Asp Hi - #s Tyr Lys Leu Ala Leu                      85  - #                90  - #                95               - - Gly Gln Ile Asn Ile Ala Lys Asn Leu Val As - #p Asn Val Ala Lys Asp                  100      - #           105      - #           110                  - - Tyr Val Arg Leu Met Lys Tyr Gly Asp Ser Le - #u Tyr Arg Cys Lys Gln              115          - #       120          - #       125                      - - Leu Lys Arg Ala Ala Leu Gly Arg Met Cys Th - #r Val Ile Lys Arg Gln          130              - #   135              - #   140                          - - Lys Gln Ser Leu Glu Tyr Leu Glu Gln Val Ar - #g Gln His Leu Ser Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Pro Thr Ile Asp Pro Asn Thr Arg Thr Le - #u Leu Leu Cys Gly        Tyr                                                                                             165  - #               170  - #               175             - - Pro Asn Val Gly Lys Ser Ser Phe Ile Asn Ly - #s Val Thr Arg Ala Asp                  180      - #           185      - #           190                  - - Val Asp Val Gln Pro Tyr Ala Phe Thr Thr Ly - #s Ser Leu Phe Val Gly              195          - #       200          - #       205                      - - His Met Asp Tyr Lys Tyr Leu Arg Trp Gln Va - #l Val Asp Thr Pro Gly          210              - #   215              - #   220                          - - Ile Leu Asp His Pro Leu Glu Asp Arg Asn Th - #r Ile Glu Met Gln Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Thr Ala Leu Ala His Leu Arg Ala Ala Va - #l Leu Tyr Val Met        Asp                                                                                             245  - #               250  - #               255             - - Leu Ser Glu Gln Cys Gly His Gly Leu Arg Gl - #u Gln Leu Glu Leu Phe                  260      - #           265      - #           270                  - - Gln Asn Ile Arg Pro Leu Phe Ile Asn Lys Pr - #o Leu Ile Val Val Ala              275          - #       280          - #       285                      - - Asn Lys Cys Asp Val Lys Arg Ile Ala Glu Le - #u Ser Glu Asp Asp Gln          290              - #   295              - #   300                          - - Lys Ile Phe Thr Asp Leu Gln Ser Glu Gly Ph - #e Pro Val Ile Glu Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Thr Leu Thr Glu Glu Gly Val Ile Lys Va - #l Lys Thr Glu Ala        Cys                                                                                             325  - #               330  - #               335             - - Asp Arg Leu Leu Ala His Arg Val Glu Thr Ly - #s Met Lys Gly Asn Lys                  340      - #           345      - #           350                  - - Val Asn Glu Val Leu Asn Arg Leu His Leu Al - #a Ile Pro Thr Arg Arg              355          - #       360          - #       365                      - - Asp Asp Lys Glu Arg Pro Pro Phe Ile Pro Gl - #u Gly Val Val Ala Arg          370              - #   375              - #   380                          - - Arg Lys Arg Met Glu Thr Glu Glu Ser Arg Ly - #s Lys Arg Glu Arg Asp      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Glu Leu Glu Met Gly Asp Asp Tyr Ile Le - #u Asp Leu Gln Lys        Tyr                                                                                             405  - #               410  - #               415             - - Trp Asp Leu Met Asn Leu Ser Glu Lys His As - #p Lys Ile Pro Glu Ile                  420      - #           425      - #           430                  - - Trp Glu Gly His Asn Ile Ala Asp Tyr Ile As - #p Pro Ala Ile Met Lys              435          - #       440          - #       445                      - - Lys Leu Glu Glu Leu Glu Lys Glu Glu Glu Le - #u Arg Thr Ala Ala Gly          450              - #   455              - #   460                          - - Glu Tyr Asp Ser Val Ser Glu Ser Glu Asp Gl - #u Glu Met Leu Glu Ile      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Arg Gln Leu Ala Lys Gln Ile Arg Glu Lys Ly - #s Lys Leu Lys Ile        Leu                                                                                             485  - #               490  - #               495             - - Glu Ser Lys Glu Lys Asn Thr Gln Gly Pro Ar - #g Met Pro Arg Thr Ala                  500      - #           505      - #           510                  - - Lys Lys Val Gln Arg Thr Val Leu Glu Lys Gl - #u Met Arg Ser Leu Gly              515          - #       520          - #       525                      - - Val Asp Met Asp Asp Lys Asp Asp Ala His Ty - #r Ala Val Gln Ala Arg          530              - #   535              - #   540                          - - Arg Ser Arg Ser Ile Thr Arg Lys Arg Lys Ar - #g Glu Asp Ser Ala Pro      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Pro Ser Ser Val Ala Arg Ser Gly Ser Cys Se - #r Arg Thr Pro Arg        Asp                                                                                             565  - #               570  - #               575             - - Val Ser Gly Leu Arg Asp Val Lys Met Val Ly - #s Lys Ala Lys Thr Met                  580      - #           585      - #           590                  - - Met Lys Asn Ala Gln Lys Lys Met Asn Arg Le - #u Gly Lys Lys Gly Glu              595          - #       600          - #       605                      - - Ala Asp Arg His Val Phe Asp Met Lys Pro Ly - #s His Leu Leu Ser Gly          610              - #   615              - #   620                          - - Lys Arg Lys Ala Gly Lys Lys Asp Arg Arg                                  625                 6 - #30                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 716 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GACTCTGCTC CCCCGTCCTC TGTGGCCCGG AGTGGGAGTT GCTCTCGAAC TC -             #CACGTGAC     60                                                                 - - GTTTCTGGTC TTAGGGATGT CAAGATGGTG AAGAAAGCCA AGACTATGAT GA -            #AGAATGCT    120                                                                 - - CAGAAGAAGA TGAATCGGTT GGGGAAGAAA GGGGAGGCGG ATATACACTT GT -            #TTGATATG    180                                                                 - - AAGCCCAAGC ACTTGCTGTC TGGGAAGAGG AAAGCTGGTA AAAAGGACAG GA -            #GATAGTAT    240                                                                 - - CCGTTTGGTT GGCGTGGCTT CGCTAGAGTG TTGCTGTTTA TTTCCTGGTT TG -            #GCACAGTA    300                                                                 - - TGGTTTCATG AAATTGGAGC TCTGTATAAA CTGAAAAAGA CAAAATAAGT AA -            #AGCACTTG    360                                                                 - - TTGCTTTGCT GAAAACTATG GTTAACCCTA TATAGGTGTG GGAAATTTTT GT -            #CACTGCAT    420                                                                 - - AATATTACAA ATATTCTGAG TAGACAGTGT TTCCACATTT AATGGAGTAT CA -            #GTTGCTTC    480                                                                 - - AGATTTTCAG AACTGGGAAG ATTTACTGGT GTAACTGGGT TGTTTTTGAT GG -            #AGAAAAAC    540                                                                 - - CTTATTTTCT TTTGTAAGAG CTGGGAGCAA ACACGTTTAT GAGTGTGTCG GA -            #ATCCCGTG    600                                                                 - - CTTAAAATAC GCTCTTAAAT TATTTTCTAG TCCTTATTTT ACAATGTCTC AT -            #TGTAGTCT    660                                                                 - - GTCTTCAACT ATTTTATCCA AAATAAACCT CCAGAAGGAA AAAAAAAAAA AA - #AAAA            716                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - His Glu Asp Lys Asp Asp Ala His Tyr Ala Va - #l Gln Ala Arg Arg Ser       1               5  - #                10  - #                15               - - Arg Ser Ile Thr Arg Lys Arg Lys Arg Glu As - #p Ser Ala Pro Pro Ser                  20      - #            25      - #            30                   - - Ser Val Ala Arg Ser Gly Ser Cys Ser Arg Th - #r Pro Arg Asp Val Ser              35          - #        40          - #        45                       - - Gly Leu Arg Asp Val Lys Met Val Lys Lys Al - #a Lys Thr Met Met Lys          50              - #    55              - #    60                           - - Asn Ala Gln Lys Lys Met Asn Arg Leu Gly Ly - #s Lys Gly Glu Ala Asp      65                  - #70                  - #75                  - #80        - - Ile His Leu Phe Asp Met Lys Pro Lys His Le - #u Leu Ser Gly Lys Arg                      85  - #                90  - #                95               - - Lys Ala Gly Lys Lys Asp Arg Arg                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ACCACACATC TGA              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TGTTGGGAAC AAG              - #                  - #                      - #      13                                                                 __________________________________________________________________________

What is claimed is:
 1. An isolated CRFG-1a polypeptide comprising anamino acid sequence which is at least 80% identical to the amino acidsequence depicted in SEQ ID NO:2 over its entire length.
 2. Thepolypeptide of claim 1 which comprises the amino acid sequence of SEQ IDNO:2.